Quality and microbial safety evaluation of new isotonic beverages upon thermal treatments

Accepted Manuscript Quality and microbial safety evaluation of new isotonic beverages upon thermal treatments Amadeo Gironés-Vilaplana, Juan-Pablo Huertas, Diego A. Moreno, Paula M. Periago, Cristina García-Viguera PII: DOI: Reference:

S0308-8146(15)01194-2 http://dx.doi.org/10.1016/j.foodchem.2015.08.011 FOCH 17956

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

4 May 2015 31 July 2015 3 August 2015

Please cite this article as: Gironés-Vilaplana, A., Huertas, J-P., Moreno, D.A., Periago, P.M., García-Viguera, C., Quality and microbial safety evaluation of new isotonic beverages upon thermal treatments, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.08.011

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Quality and microbial safety evaluation of new isotonic beverages upon thermal treatments Amadeo Gironés-Vilaplanaa, Juan-Pablo Huertasb, Diego A. Moreno a, Paula M. Periago b, Cristina García-Vigueraa*

a

CEBAS-CSIC Phytochemistry Lab. Department of Food Science and Technology,

P.O. Box 164, E-30100, Espinardo, Murcia, Spain. b

Departamento de Ingeniería de Alimentos y del Equipamiento Agrícola, Campus de

Excelencia Internacional Regional "Campus Mare Nostrum", Universidad Politécnica de Cartagena, Paseo Alfonso XIII 48, 30203 Cartagena, Murcia, Spain.

*Corresponding author: Cristina García Viguera CEBAS-CSIC Food Science and Technology Department P.O.Box 164, E-30100, Espinardo, Murcia, Spain. Tel +34 968 396304; fax +34 968 396213; E-mail: [email protected]

Running title: Influence of heat treatments on isotonic drink during shelf life

1

1

Abstract

2

In the present study, it was evaluated how two different thermal treatments (Mild and

3

Severe) may affect the anthocyanin content, antioxidant capacity (ABTS+, DPPH•, and

4

FRAP), quality (CIELAB colour parameters), and microbiological safety of a new

5

isotonic drink made of lemon and maqui berry over a commercial storage simulation

6

using a shelf life of 56 days at two preservation temperature (7°C and 37°C). Both heat

7

treatments did not affect drastically the anthocyanins content and their percentage of

8

retention. The antioxidant capacity, probably because of the short time, was also not

9

affected. The CIELAB colour parameters were affected by the heat, although the

10

isotonic drinks remained with attractive red colour during shelf life. From a

11

microbiological point of view, the Mild heat treatment with storage at 7ºC is the ideal

12

for the preservation of microbial growth, being useful for keeping the quality and safety

13

of beverages in commercial life.

14

15

Keywords: Heat treatment, microbiology, anthocyanins, bioactivity, shelf life

16

2

17

1. Introduction

18

The design of new fruit beverages, rich in bioavailable and bioactive compounds,

19

can be the basis of new functional foods with potential health benefits, due to the close

20

relationship between a physiological positive state of oxidative stress as a trigger for

21

different health problems (cardiovascular, metabolism of glucose and lipids, neuronal

22

activity, anxiety, etc.) (Packer, Cadenas, & Davies, 2008). The growing interest in new

23

products with health-promoting properties has led to the development of new beverages

24

based on fruit juices, as source of nutrients and bioactive compounds, being an

25

important portion of the global functional markets (Leatherhead Food Research 2011).

26

In this sense, new isotonic drinks made with lemon juice (Citrus limon (L.) Burm. f.)

27

and maqui berry (Aristotelia chilensis) were made in previous research, obtaining

28

interesting phytochemical composition, biological activity, enzyme modulation

29

capacity, and organoleptic acceptability (Gironés-Vilaplana, Mena, Moreno, & García-

30

Viguera, 2013; Gironés-Vilaplana, Villaño, Moreno, & García-Viguera, 2013), but with

31

the idea in mind of the elaboration of the drink at the industry level, the protocols and

32

conditions for quality and safety during shelf life needs to be established, for the future

33

commercialization of these beverages.

34

The demand by consumers for “natural” drinks has led to the use of non-

35

aggressive technologies, being refrigeration the most common perseveration technique

36

for many drinks and fruit juices. However, the difficulty in maintaining the cold-chain

37

throughout the production, distribution and storage, implies the use of additional

38

barriers (or hurdles) to control spoilage and microorganisms. Commercial fruit

39

beverages, generally, are pasteurized at temperatures between 85ºC and 95ºC for a few

40

minutes or seconds. This thermal treatment is a relative Mild heat treatment which 3

41

inactivates non-spore forming micro-organisms (pathogens, lactic acid bacteria, yeasts

42

and moulds), which could spoil these products, while the germination and growth of the

43

surviving bacterial spores are inhibited by the stressful product conditions (pH < 4)

44

(Bevilacqua, Sinigaglia, & Corbo, 2009). Thermal treatments applied in acidic foods are

45

used to extend shelf life for several months by destruction of spoilage microorganisms

46

(yeast and moulds) and or enzyme inactivation, being the minimum processing

47

conditions 65ºC for 30 min, 77ºC for 1 min or 88ºC for 15 s (Fellows, 2000). Thermal

48

treatments cause important losses in sensorial and nutritional properties, making such

49

alternative unviable to preserve natural fresh-like juices (Bevilacqua, et al., 2009).

50

Hurdle technology consists of the use of combined preservative factors (i.e.

51

temperature, water activity, pH) for gentle, but effective, conservation of a variety of

52

foods. This concept was developed by Leistener (Leistener & Gould, 2005). As an

53

example, a synergistic effect of heat with low-pH that allow mild heating to deliver

54

ambient stability of acidic foods has been showed (Alakomi, Skyttä, Helander, &

55

Ahvenainen, 2002). Despite this, certain microorganisms may survive at low-pH, during

56

processing and storage and may cause spoilage in fruit beverages (Huertas, Esteban,

57

Antolinos, & Palop, 2013; Parish, 2009).

58

Historically, citrus beverages were not considered to be associated with a high

59

risk for causing foodborne illness. The pH and the organic acid content of citrus drinks

60

was a challenge for the survival or growth of bacteria, coupled with a high sugar

61

content, resulting in a microbiological population made up primarily of acidolactic

62

bacteria, yeasts, and moulds (Keller & Miller, 2006; Parish, 2009).

63

In order to assure the microbiological safety and stability of healthy foods, such as

64

fruit beverages (rich in bioavailable and bioactive compounds), it is necessary to apply 4

65

balanced hurdles (i.e. pH, heat treatment, storage conditions), in a more complex way,

66

ensuring product safety and stability, achieving an hostile environment to inhibit the

67

growth, shorten the survival or killing them, while not damaging the product’s sensory

68

and nutritional properties (Alakomi et al, 2002). In this way, we can answer to

69

consumer demands for healthier and tasty foods.

70

Therefore, the aim of this work was to evaluate how the heat treatments (Mild and

71

Severe) may influence the anthocyanin composition, the antioxidant capacity, quality,

72

and the microbiological safety of this isotonic drink made of lemon juice and maqui

73

berry during shelf life (56 days) at two temperatures (7ºC and 37ºC), compared to

74

controls untreated, to gather the necessary information to recommend to the industry for

75

the further elaboration of this beverage for the markets.

76

5

77

2. Material and Methods

78

2.1. Chemicals

79

The compounds 2,2-diphenyl-1-picrylhidracyl radical (DPPH•), 2,2-azino-bis(3-

80

ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS˙+), 2,4,6-tripyridyl-S’-

81

triazine (TPTZ), ferric chloride hexahydrate, and potassium phosphate were obtained

82

from

83

tetramethylchroman-2-carboxylic acid (Trolox), and magnesium chloride hexahydrate

84

were purchased from Fluka Chemika (Neu-Ulm, Switzerland); sodium carbonate

85

(anhydrous), sodium benzoate, and potassium sorbate were bought from Panreac

86

Química S.A. (Barcelona, Spain). Ultrapure water was produced using a Millipore water

87

purification system.

88

2.2. Isotonic drinks design

Sigma-Aldrich

(Steinheim,

Germany).

Meanwhile,

6-hydroxy-2,5,7,8-

89

New isotonic beverages composition for 100 mL was: 80 mL of water, 20 mL of

90

lemon juice (pH: 2.37, TA: 5.4%), 7.5 g (w/v) of sucrose, 5 g of maqui lyophilized

91

berry, 20 mg of NaCl, 6 mg of Potassium phosphate, and 33 mg of potassium sorbate

92

and 16 mg of sodium benzoate as conservants (according to Spanish regulation: RD

93

142/2002). Simultaneously, one control was also designed using the same ingredients

94

except 20 mL of citric acid solution 5g/100 mL (pH: 2.31, TA: 5.1%) instead of lemon

95

juice. Samples were filtrated later by cheesecloth from Texpol (Barcelona, Spain).

96

Samples were labelled as follows: LM (isotonic drink of lemon juice plus maqui

97

berry), and IM (isotonic drink of citric acid plus maqui berry, as control). Analyses were

98

carried out in triplicates every 14 days during all preservation study.

99

2.3. Thermal treatments and microbiological determinations

6

100

Thermal treatments were carried out in a thermoresistometer Mastia (Conesa,

101

Andreu, Fernández, Esnoz, & Palop, 2009). The vessel of the instrument was sterilized

102

before the treatments, for this the vessel was filled with distilled water and heated at

103

135ºC for 2 minutes, then cooled, emptied and immediately (in sterile conditions) filled

104

with 400 mL of the isotonic drinks. All treatments started at a temperature of 25ºC. For

105

the Mild treatment the thermoresistometer was programmed to reach a final temperature

106

of 80 ºC with a heating rate of 30 ºC/min, and when the sample reached the final

107

temperature it was cooled immediately to a final temperature of 40ºC at cooling rate of

108

30ºC/min. For the Severe treatment the equipment was programmed to reach a final

109

temperature of 85 ºC with a heating rate of 30 ºC/min, holding the temperature for 6

110

seconds, and immediately cooled down to a final temperature of 40ºC at 30ºC/min.

111

Samples of the isotonic drinks were taken in sterile Falcon tubes during the process. For

112

the Mild treatment, samples were taken at the following process temperatures: 25, 70,

113

80 and 40ºC. For the Severe treatment, samples were taken at process temperatures of

114

25ºC, when the product reached the final temperature process (85ºC), after the holding

115

phase (6 s at 85ºC) and at 40ºC. Samples were stored at 7ºC and at 37ºC.

116

For microbiological tests, samples before treatments (control) and after treatments (heat

117

treated) were analyzed for psycrophilic and mesophilic microorganisms, yeasts, and

118

moulds. For viable plate counts of mesophilic and psycrophilic bacteria, Plate Count

119

Agar (PCA) (Sharlab, Barcelona) was used, and incubated for 24 h at 37ºC and for 1

120

week at 7ºC, respectively. Yeasts and moulds were plated on Rose Bengal Agar

121

(Sharlab, Barcelona) and incubated at 25ºC for 1 week (after incubation time plates

122

were counted).

123

2.4. pH and Total Soluble Solids (TSS) 7

124

pH, and Total Soluble Solids (TSS) were evaluated as quality indexes following

125

the method reported by Mena et al. (P. Mena, Gironés-Vilaplana, Martí, & García-

126

Viguera, 2012). Results were expressed as ºBrix in TSS.

127

2.5. Identification of anthocyanins by HPLC-DAD-ESI/MS n, and quantification and

128

evolution by RP-HPLC-DAD

129

The anthocyanins from maqui berry were identified in previous works (Gironés-

130

Vilaplana, Baenas, Villaño, Speisky, García-Viguera, & Moreno, 2014; Gironés-

131

Vilaplana, Mena, García-Viguera, & Moreno, 2012; Gironés-Vilaplana, Mena, et al.,

132

2013), and were confirmed by HPLC-DAD-ESI-MSn analysis. Chromatographic

133

analyses for the identification were carried out on a Luna C18 column (250 x 4.6 mm, 5

134

mm particle size; Phenomenex, Macclesfield, UK). Water:formic acid (99:1, v/v) in an

135

Agilent HPLC 1100 series equipped with a photodiode array detector and a mass

136

detector in series (Agilent Technologies, Waldbronn, Germany) with the same

137

conditions used previously according to Gironés-Vilaplana et al.. (Gironés-Vilaplana,

138

Mena, et al., 2013). The equipment consisted of a binary pump (model G1312A), an

139

autosampler (model G1313A), a degasser (model G1322A) and a photodiode array

140

detector (model G1315B). The HPLC system was controlled by ChemStation software

141

(Agilent, version 08.03)

142

For the quantification all samples were centrifuged for 5 min at 10500 rpm. Each

143

supernatant was filtered through a 0.45-µm PVDF filter (Millex HV13, Millipore,

144

Bedford, MA, USA) before injection into the HPLC system, as described by Gironés-

145

Vilaplana et al. (Gironés-Vilaplana, Villaño, et al., 2013). Chromatograms were

146

recorded at 520 nm, and anthocyanins were quantified as cyanidin 3-O-glucoside.

147

2.6. Antioxidant capacity 8

148

The free radical scavenging activities were determined using the DPPH•, ABTS˙+,

149

and FRAP (ferric reducing antioxidant power) methods adapted to a microscale,

150

according to Mena et al. (2011) (Pedro Mena, et al., 2011). The antioxidant activity was

151

evaluated by measuring the variation in absorbance at 515 nm after 50 min of reaction

152

with the radical (for DPPH•), at 414 nm after 50 min (ABTS˙+), and at 593 nm after 40

153

min for FRAP. The assays were performed using 96-well micro plates (Nunc, Roskilde,

154

Denmark) and an Infinite M200 micro plate reader (Tecan, Grödig, Austria). All the

155

reactions were started by adding 2 µL of the corresponding diluted sample to the well

156

containing the stock solution (250 µL). The final volume of the assay was 252 µL, and

157

the results were expressed as mM Trolox.

158

2.7. Colour measurements

159

Solutions were measured in glass cells of 10-mm path length (CT-A21) at 520 nm

160

using a Minolta CM-508i® tristimulus colour spectrophotometer (Osaka, Japan) coupled

161

with a CM-A760 transmittance adapter. CIEL∗, a∗ and b∗ values were calculated using

162

illuminant D65 and a 10◦ observer, according to the CIEL∗ a ∗ b ∗ 76 Convention

163

(McLaren, 1980). Data were recorded and processed on a Minolta Software

164

ChromaControl S, PC-based colorimetric data system. Hue angle (H) was calculated

165

from tan−1 (b*/a*) and Chroma (C*) from (a*2 + b*2)1/2. Colour difference were also

166

calculated: ∆E*= [(∆L*)2 + (∆a*)2 +(∆b*)2]1/2, taking the day 0 of both liquors as a

167

reference. All measurements were done in triplicate, and the mean values reported in

168

each case.

169

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3. Results and Discussion

171

3.1. Quality parameters

172

The quality parameters showed slight differences between isotonic drinks, being

173

pH and soluble solids (SS) of LM lower than their respective control (IM). These values

174

were not different during the 4 points of each treatment. Moreover, quality parameters

175

did not show significant differences during the storage of 56 days, although differences

176

were observed between LM treated (Severe and Mild) and LM untreated in SS, which

177

increased over shelf life. It is important to note that all parameters were within the

178

normal and acceptable range for these kinds of drinks (Jain, Hall-May, Golabek, &

179

Agustin, 2012).

180

3.2. Anthocyanins content and retention during shelf life

181

Regarding anthocyanins, those described previously for maqui were identified,

182

with a total amount in the beverages of ~ 38 mg/100 mL in LM, and ~ 33 mg/100 mL in

183

IM (Gironés-Vilaplana, et al., 2014; Gironés-Vilaplana, Mena, et al., 2012) (Figure 1A).

184

No significant reductions in total and individual quantities were recorded during heating

185

indistinctly of the thermal treatment used: in Mild treatment no changes in the total

186

anthocyanins content was observed during the 4-point sampling, in contrast what

187

happened in Severe treatment, in which the anthocyanins content increased during

188

heating (Figure 1B). This may be due to the consequence of native copigments and self-

189

association of anthocyanins that improve color stability of the juices (Brenes, Del Pozo-

190

Insfran, & Talcott, 2005). The conversion of leucoanthocyanin to anthocyanin could

191

also happen, but probably at a slower rate of thermal degradation, as reported Lee et al.

192

(Lee, Durst, & Wrolstad, 2002) for pasteurized blueberry juice with higher amounts of 10

193

anthocyanins than initial pressed juice, and in pomegranate juices with increased

194

anthocyanins after thermal treatment (Alighourchi, Barzegar, & Abbasi, 2008).

195

It is widely known that the stability of anthocyanins is influenced during storage

196

by multiple factors including chemical structure, pH, temperature, enzymes, oxygen,

197

light, ascorbic acid, sugars, metals, and copigments (Castañeda-Ovando, Pacheco-

198

Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal, 2009; Francis, 1989). As

199

expected, the samples stored at 7ºC preserved better the anthocyanin content compared

200

to those stored at 37°C, retaining more than 50% of initial anthocyanin content at the

201

end of study period (Figure 1C and Figure 1D). No remarkable differences in

202

anthocyanin retention rate over time were observed, concluding that heat treatments did

203

not affect negatively the anthocyanin content in the beverages. The lack of an expected

204

reduction of concentration of anthocyanins after heating could be due to the short times

205

used in the treatments, which resulted in more efficient thermal processes than other

206

reported (Buckow, Kastell, Terefe, & Versteeg, 2010; Li, Song, Dong, & Zhao, 2014).

207

Moreover, the shelf life values of anthocyanins obtained were considerably higher than

208

those reported for other thermally pasteurized berry juices (Alighourchi, et al., 2008;

209

Gössinger, et al., 2009). This fact emphasizes the need for developing novel approaches

210

avoiding long-time thermal treatments to limit microbial loads of juices and to preserve

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as much as possible the anthocyanins content of berry juices. Nevertheless, isotonic

212

beverages made with lemon juice showed higher anthocyanin degradation than controls

213

with citric acid, probably due to the presence of ascorbic acid from the lemon juice,

214

which has proven to lead to the breakdown of anthocyanins (Gironés-Vilaplana, Mena,

215

et al., 2012; Gironés-Vilaplana, Mena, et al., 2013).

216

3.3. Antioxidant activity 11

217

The antioxidant activity was much higher in LM than in IM drinks in the 3

218

antioxidant capacity assays used: ABTS+, DPPH˙, and FRAP, probably due to the

219

presence of lemon juice (Figure 2). With respect to ABTS+, similar high values were

220

obtained than those recently published (Gironés-Vilaplana, Mena, et al., 2013),

221

supporting the strong in vitro antioxidant capacity of maqui in this assay, attributed to

222

its polyphenolic content (Céspedes, El-Hafidi, Pavon, & Alarcon, 2008; Miranda-

223

Rottmann, Aspillaga, Pérez, Vasquez, Martinez, & Leighton, 2002). Few losses were

224

noted in the antioxidant capacity of beverages during storage, slightly higher in those

225

stored at 37ºC, being correlated to the anthocyanin degradation in all samples (p<0.01).

226

However, in contrast to what happened with anthocyanin content, when the degradation

227

or loss rate was similar in all isotonic drinks, in this method the beverages not heated

228

showed a slightly higher antioxidant capacity than samples subjected to heat treatments,

229

probably due to other components responsible for this antioxidant activity that can be

230

degraded during thermal heats, such as vitamin C among others, with demonstrated low

231

stability during thermal processes (Vega-Gálvez, et al., 2009).

232

Regarding DPPH• method, lower antioxidant capacity values were obtained, as

233

expected since samples were aqueous, and DPPH• is normally used with methanolic

234

extracts, being in similar range than previously reported data of maqui extracts and

235

lemon-maqui blends (Céspedes, et al., 2008; Gironés-Vilaplana, Valentão, Moreno,

236

Ferreres, García-Viguera, & Andrade, 2012; Gironés-Vilaplana, Villaño, et al., 2013).

237

The degradation of anthocyanins was also correlated to antioxidant activity losses (p <

238

0.001), but not in all isotonic drinks (not correlated for: untreated LM and IM stored at

239

7ºC, IM untreated stored at 37ºC, and Mild treated IM stored at 7ºC and 37ºC). All the

240

samples showed more stable results than in the ABTS+ in shelf-life. Although the 12

241

untreated isotonic drinks retained a higher antioxidant capacity than beverages with heat

242

treatments applied (Figure 2). The phytochemical losses during thermal treatments

243

explained before, such as vitamin C among others, could be the reason, since ascorbic

244

acid is commonly used as equivalent to express the antioxidant capacity in ABTS+ and

245

DPPH• methods, due to its reactivity against both radicals (Kim, Lee, Lee, & Lee,

246

2002).

247

The great FRAP scavenging capacity showed was previously reported for

248

lyophilized maqui berries (Gironés-Vilaplana, et al., 2014) and an alcoholic maqui

249

liquor (Gironés-Vilaplana, Calín-Sánchez, Moreno, Carbonell-Barrachina, & García-

250

Viguera, 2015), attributed to its anthocyanin contents (Figure 2). As in the DPPH•

251

method, the antioxidant capacity losses were also correlated to the degradation of

252

anthocyanins (p < 0.001), but not in all isotonic drinks (not correlated for: untreated LM

253

stored at 7ºC and 37ºC, untreated IM stored at 7ºC, Mild treated IM stored at 7ºC, and

254

Severe treated IM stored at 7ºC and 37ºC). In the FRAP method the degradation of

255

antioxidant capacity was lower in all samples, except for untreated LM stored at 7ºC

256

and 37ºC, although this result was higher in these non-treated drinks at the beginning of

257

the study (Figure 2). These slight differences could be explained by the different

258

mechanisms involved in FRAP assay, where antioxidants react against Fe3+-TPTZ

259

complex (Benzie & Strain, 1996).

260

3.4. Colour: CIELAB parameters

261

The newly designed isotonic beverages with natural anthocyanins from maqui

262

give an attractive red/dark and natural colour for consumer acceptance. For this reason,

263

colour parameters were determined and studied during both heat treatments and over the

264

56 days of shelf-life simulation. Regarding both thermal treatments, Severe heating 13

265

displayed more changes in all CIEL*a*b* parameters than Mild treatment (p<0.05),

266

although these changes did not affect significantly the colour of the new isotonic drink

267

(Table 1).

268

Is important to emphasize that both isotonic drinks (IM and LM) suffered

269

significant changes between the end of the thermal treatments and day 14 of storage,

270

inducing that the major changes in the CIEL*a*b * parameters occurred at the end of the

271

heating treatments, not during them (Table 2). With respect to CIEL* values, samples

272

stored at 7ºC did not changed since day 14, while isotonic beverages (IM and LM)

273

stored at 37ºC showed an appreciable increase in this parameter, with strong negative

274

correlations between CIEL* and total anthocyanin content degradation (LM untreated (r

275

= -0.846, p = 0.001), IM untreated (r = -0.947, p < 0.001), Mild LM (r = -0.834, p =

276

0.001), Mild IM (r = -0.971, p < 0.001), Severe LM (r = -0.958, p < 0.001), and Severe

277

IM (r = -0.993, p < 0.001)). This fact suggests that the anthocyanins degradation was

278

related with the increased lightness (or decrease of darkness) over the shelf-life, as

279

demonstrated before for this kind of blends (Gironés-Vilaplana, Mena, et al., 2013). In

280

beverages stored at 7ºC, CIEa* values remained quite stable after the significant change

281

that occurred at the end of thermal treatments. In samples stored at 37ºC, this parameter

282

decreased in the 56 days studied, and was strongly correlated to the total anthocyanins

283

degradation (LM untreated (r = 0.781, p < 0.01), IM untreated (r = 0.984, p < 0.001),

284

Mild LM (r = 0.840, p = 0.001), Mild IM (r = 0.965, p < 0.001), Severe LM (r = 0.966,

285

p < 0.001), and Severe IM (r = 0.994, p < 0.001)), which was indicative of the influence

286

of anthocyanins in the redness of the product. The same thing happened for CIEb*

287

parameters at 7ºC, while at 37ºC an increase was observed in all isotonic drinks,

288

negatively and strongly correlated to anthocyanins degradation (LM W.T. (r = -0.820, p 14

289

= 0.001), IM W.T. (r = -0.957, p < 0.001), LM Mild (r = -0.750, p < 0.01), IM Mild (r =

290

-0.947, p < 0.001), LM Severe (r = -0.869, p < 0.001), and IM Severe (r = -0.983, p <

291

0.001)), probably due to the conversion of red anthocyanin to the yellow chalcone, since

292

CIEb* is associated to yellowness. The Chroma value is related to CIEa* and CIEb*,

293

although remained more stable than these two parameters in all samples, without

294

notable differences between heat treatments during storage. Regarding Hue angle, no

295

significant changes were reported at 7ºC, showing an increase at 37ºC during storage

296

period, as in the rest of colour parameters.

297

It is important to note, that regardless of anthocyanin losses during shelf life, the

298

red coloration of all the isotonic drinks with the different thermal treatments remained

299

quite stable during the 56 days of storage, as a result of the likely formation of other

300

coloured-polymers (Boulton, 2001), or copigmentation between anthocyanins and other

301

flavonoids that appreciably preserved colour and masked the detrimental changes during

302

storage in the anthocyanins (Brenes, et al., 2005).

303

3.5. Microbiology

304

The IM did not shown any microbiological growth during shelf life time at the

305

different conditions tested, not even in the non-heat treated samples. The absence of

306

growth in IM samples, even the non-thermal treated ones, could be due the absence of

307

native microflora adapted to the characteristics and conditions of the product (i.e. pH,

308

nutrients). Therefore, the product characteristics (such as pH) could be restrictive for the

309

microbial flora contamination derived from the manipulation and processing, preventing

310

the outgrowth of contaminants and making the product stable during the storage under

311

abuse temperature conditions (37ºC) even without a thermal treatment.

15

312

Tables 3 and 4 show microbiological results for LM analyzed during the shelf life

313

during its storage at refrigeration temperature (7ºC) or under abuse temperature (37ºC).

314

For LM samples stored at refrigeration temperature, the non-heat treated samples

315

presented growth of yeasts & moulds from day 14, and of psycrotrophic bacteria from

316

day 28 of storage (Table 3). Mesophilic bacteria from samples kept at 7ºC did not

317

present any growth (< 10 CFU/mL) during the whole storage (56 days). LM non-heat

318

treated samples kept at refrigeration temperature showed a wide diversity of yeasts &

319

moulds, and its growth was prior to bacterial growth. The growth of yeasts & moulds

320

could lead to changes in the product characteristics allowing the subsequent growth of

321

bacteria (i.e: pH, solubilization of nutrients, etc.). This could be due to a positive

322

relationship (commensalism) between microbial populations, in which a population

323

(bacteria) benefits by the former growth of another population (yeasts & moulds)(Atlas

324

& Bartha, 1997; Gram, Ravn, Rasch, Bruhn, Christensen, & Givskov, 2002; Viljoen,

325

2001). Remarkably, Severe or Mild heat treated samples kept at refrigeration

326

temperature, did not present growth of yeasts & moulds nor of bacteria, during the days

327

of the study (Table 3).

328

Under abuse temperature storage (30ºC), no bacterial growth was found in LM

329

samples after 56 days (Table 4). However, LM samples presented growth only for one

330

species of mould (identification were not done), which was detected at day 28 for non

331

heat-treated samples, and in the last day (56 days) of the storage time, for the heat-

332

treated samples (Table 4). As described in material and methods section, the lemon

333

juice used for the LM preparation was done in non-sterile conditions. It is well known

334

that native microflora from products is well adapted to the characteristics of the product

335

(i.e. pH, nutrients, antimicrobial compounds) and its outgrowth is easy for this reason. It

336

could be possible that native micro-flora from the peel of the lemon has been transferred 16

337

to the lemon juice during its extraction, and these adapted microorganisms were able to

338

outgrowth and spoilage the product.

339 340

Therefore, from a microbiological point of view, the LM product would need a Mild thermal treatment and storage at 7ºC for a longer shelf life.

341

17

342

4. Conclusions

343

It has been determined that the thermal treatments used did not affect drastically

344

the anthocyanins content and retention and the antioxidant capacity of the isotonic

345

drinks, probably due to the short time used in the treatments. Although CIEL*a*b*

346

colour parameters were affected by heating, beverages remained with attractive and red

347

coloration during the 56 days of storage, probably due to copigmentation reactions.

348

Moreover, from a microbiological point of view, the softer heat treatment (Mild), with

349

preservation at 7ºC, is the ideal to prevent microbial growth while keeping the quality

350

and bioactivity of the isotonic drink. Therefore, it can be concluded that the LM drinks,

351

with higher antioxidant capacity, with Mild treatment applied and kept at 7°C displayed

352

high bioactivity, a significant amount of phytochemicals and guaranteed the

353

microbiological safety during storage, which is useful for commercialization of these

354

beverages.

355

18

356

Acknowledgments

357

The authors express their gratitude to the Spanish Ministry of Economy and

358

Competitiveness for the CYTED Program (Ref. 112RT0460) CORNUCOPIA Thematic

359

Network (URL: redcornucopia.org) and for the FEDER (Fondo Europeo de Desarrollo

360

Regional) (Ref. AGL 2013/48993-C2-1-R). AGV thanks the CSIC and the European

361

Social Fund for a JAE pre-doctoral grant. Juan Pablo Huertas also is grateful to the

362

Spanish Ministry of Science and Innovation for the concession of pre-doctoral grant

363

(BES-2011-046580).

364

19

365 366

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23

476 477

Figure captions

478

Figure 1. Anthocyanin profile and content (mg/100 mL) of both isotonic drinks (AM

479

and LM) within 4-point heat treatments (Mild, Severe), and % of retention during shelf

480

life at the two temperatures studied (7°C and 37°C). In Figure 2B, n=3 ± SD, and

481

different letters means significantly different at P < 0.05 according to Tukey HSD

482

Multiple Range Test.

483

Figure 2. Antioxidant activity (ABTS+, DPPH˙, and FRAP) of both isotonic (AM and

484

LM) during the 4-point heat treatments (A), and over the shelf life (B) at both

485

temperatures studied: 7°C and 37°C. N=3 ± SD, and different letters means significantly

486

different at P < 0.05 according to Tukey HSD Multiple Range Test.

487

24

Fig. 1

A1+A2: Delphinidin 3-O-sambubioside-5-O-glucoside + Delphinidin 3,5-O-diglucoside (coeluted), A3+A4: Cyanidin 3,5-O-diglucoside + Cyanidin 3-Osambubioside-5-O-glucoside (coeluted), A5: Delphinidin 3-O-sambubioside, A6: Delphinidin 3-O-glucoside, A7: Cyanidin 3-O-sambubioside, and A8: Cyanidin 3-O-glucoside-5-O-rhamnoside.

25

Fig. 2

26

Table 1. CIELAB colour parameters of both isotonic drinks during the four thermal points of the different heat treatments (Mild and Severe). Lemon + Maqui (LM) T.Points 1 12.45 ± 0.1 b L* 39.57 ± 0.4 b a* 21.31 ± 0.2 b b* 44.94 ± 0.4 b Chroma 28.31 ± 0.0 bc Hue Citric acid + Maqui (IM) T.Points 1 35.37 ± 1.7 b L* 55.44 ± 1.1 b a* 28.17 ± 3.5 ab b* Chroma 62.24 ± 0.6 ab 26.92 ± 3.6 b Hue

MILD 2 13.61 ± 0.3 b 40.88 ± 0.6 b 23.30 ± 0.4 b 47.06 ± 0.7 b 29.68 ± 0.1 bc MILD 2 32.96 ± 0.8 a 53.91 ± 0.8 a 38.08 ± 4.5 d 66.07 ± 2.0 c 35.18 ± 3.6 cd

3 13.39 ± 0.4 b 40.63 ± 0.8 b 22.92 ± 0.7 b 46.65 ± 1.0 b 29.42 ± 0.3 bc

4 12.45 ± 0.1 b 39.54 ± 0.4 b 21.30 ± 0.1 b 44.91 ± 0.4 b 28.32 ± 0.1 bc

1 12.20 ± 0.5 b 38.97 ± 0.9 b 20.62 ± 1.2 b 43.99 ± 1.5 b 26.34 ± 3.0 b

3 32.99 ± 0.2 a 53.86 ± 0.1 a 38.00 ± 0.8 d 65.92 ± 0.4 c 35.20 ± 0.6 cd

4 35.82 ± 1.6 bc 55.40 ± 0.7 b 28.63 ± 4.0 b 62.42 ± 1.2 ab 27.30 ± 3.5 b

1 37.62 ± 0.1 c 56.65 ± 0.7 c 22.30 ± 1.2 a 60.89 ± 1.1 a 21.49 ± 0.8 a

SEVERE 2 6.34 ± 1.5 a 31.68 ± 3.2 a 10.76 ± 2.5 a 33.48 ± 3.8 a 18.64 ± 2.3 a SEVERE 2 33.35 ± 0.1 a 53.69 ± 0.3 a 35.39 ± 0.4 cd 64.30 ± 0.5 bc 33.39 ± 0.2 cd

N=3 ± SD. Different letters means significantly different at P < 0.05 according to Tukey HSD Multiple Range Test.

27

LSD P<0.05

3 5.94 ± 0.8 a 30.99 ± 1.9 a 10.08 ± 1.4 a 32.59 ± 2.2 a 17.98 ± 1.3 a

4 6.28 ± 1.4 a 31.59 ± 3.0 a 10.67 ± 2.4 a 33.36 ± 3.7 a 18.55 ± 2.2 a

0.465 1.026 0.821 1.247 0.937

3 32.80 ± 0.8 a 53.28 ± 0.4 a 38.84 ± 4.0 d 65.97 ± 2.1 c 36.05 ± 3.0 d

4 34.46 ± 0.5 ab 54.16 ± 0.2 a 31.89 ± 1.8 bc 62.86 ± 1.0 ab 30.48 ± 1.4 bc

0.543 0.350 1.710 0.734 1.417

Table 2. CIELAB colour parameters of isotonic drinks (IM and LM) over the shelf life at both temperatures (7°C and 37°C) after all thermal treatments (Untreated, Mild and Severe). Lemon + Maqui (LM) Days 14 25.53 ± 1.8 a L* 55.81 ± 1.6 d a* 43.86 ± 3.1 a b* 70.99 ± 3.1 a Chroma 38.14 ± 1.2 b Hue Days L* a* b* Chroma Hue

14 38.32 ± 12 a 63.20 ± 0.3 d 41.66 ± 7.3 d 75.83 ± 3.8 b 33.28 ± 4.7 ab

Days 14 33.99 ± 1.1 a L* 60.28 ± 0.3 e a* 57.45 ± 0.5 e b* 83.27 ± 0.6 b Chroma 43.62 ± 0.1 a Hue Citric acid + Maqui (IM) Days 14 48.60 ± 2.4 a L* 65.02 ± 0.8 e a*

UNTREATED 7ºC 28 42 26.09 ± 2.3 a 26.83 ± 2.2 a 55.59 ± 2.1 d 56.06 ± 2.0 d 44.84 ± 4.0 a 46.10 ± 3.8 a 71.43 ± 4.1 a 72.59 ± 4.0 a 38.85 ± 1.5 b 39.39 ± 1.4 b MILD 7ºC 28 42 39.88 ± 1.7 a 40.05 ± 1.5 a 63.61 ± 0.7 d 63.41 ± 0.5 d 41.48 ± 1.6 d 39.43 ± 0.9 bc 73.53 ± 3.7 b 72.34 ± 3.3 b 29.68 ± 6.2 a 28.38 ± 5.7 a SEVERE 7ºC 28 42 34.50 ± 1.2 ab 35.47 ± 0.7 ab 59.82 ± 0.3 e 60.11 ± 0.2 e 56.82 ± 1.5 e 56.71 ± 4.1 e 82.52 ± 0.9 b 82.68 ± 3.0 b 43.52 ± 0.9 a 43.31 ± 2.0 a UNTREATED 7ºC 28 42 48.99 ± 2.1 a 50.89 ± 0.2 a 64.37 ± 0.7 e 63.16 ± 0.3 e

56 27.43 ± 0.1 a 55.50 ± 1.5 d 46.10 ± 1.3 a 71.70 ± 2.6 a 38.87 ± 1.2 b

14 35.35 ± 1.8 b 56.91 ± 0.4 d 60.80 ± 3.1 b 83.29 ± 2.0 b 46.87 ± 1.7 b

56 41.29 ± 1.8 a 63.35 ± 0.2 d 31.41 ± 7.1 ab 70.86 ± 3.0 b 26.26 ± 5.2 a

14 56.41 ± 2.2 b 34.28 ± 3.2 c 28.46 ± 1.8 a 44.57 ± 3.6 a 39.74 ± 0.9 b

56 36.52 ± 1.5 b 60.12 ± 0.6 b 53.48 ± 8.6 de 80.58 ± 5.3 b 41.47 ± 4.9 a

14 49.53 ± 1.7 c 40.74 ± 3.4 d 34.16 ± 1.9 a 53.16 ± 3.9 a 40.01 ± 0.8 a

UNTREATED 37ºC 28 42 40.80 ± 3.0 c 45.89 ± 1.4 d 49.76 ± 2.1 c 37.14 ± 0.1 b 68.51 ± 2.8 c 71.84 ± 0.2 c 84.70 ± 1.0 b 81.40 ± 1.0 b 54.00 ± 2.3 b 63.43 ± 1.1 b MILD 37ºC 28 42 64.12 ± 1.9 c 65.77 ± 1.7 c 20.55 ± 2.1 b 15.61 ± 1.6 a 37.14 ± 2.7 bc 42.76 ± 3.5 d 42.45 ± 3.4 a 45.52 ± 3.9 a 61.07 ± 0.7 c 69.96 ± 0.4 d SEVERE 37ºC 28 42 56.68 ± 1.8 d 60.52 ± 0.3 e 23.55 ± 1.7 c 17.82 ± 0.5 b 43.80 ± 2.5 b 47.67 ± 0.7 cd 49.73 ± 3.1 a 50.89 ± 0.8 a 61.75 ± 0.4 b 69.52 ± 0.3 c

14 57.58 ± 3.4 b 44.23 ± 2.8 d

UNTREATED 37ºC 28 42 56 65.73 ± 1.9 c 68.29 ± 2.3 cd 72.05 ± 2.6 d 27.14 ± 2.1 c 19.04 ± 2.5 b 14.11 ± 2.1 a

56 49.44 ± 2.2 a 63.76 ± 1.3 e 28

LSD P<0.05 56 48.38 ± 0.8 d 33.62 ± 0.2 a 79.70 ± 1.8 d 86.23 ± 1.2 b 67.06 ± 0.5 b 56 66.88 ± 1.4 c 13.52 ± 1.6 a 44.71 ± 3.5 d 44.21 ± 0.3 a 73.20 ± 0.7 d 56 63.00 ± 0.4 f 14.86 ± 1.1 a 48.26 ± 2.0 cd 50.50 ± 2.2 a 72.90 ± 0.5 c

1.093 0.858 1.620 1.540 0.827 LSD P<0.05 0.983 0.928 2.437 1.911 2.257 LSD P<0.05 0.712 0.835 2.114 1.684 1.106 LSD P<0.05 1.414 1.167

b* Chroma Hue

8.75 ± 2.9 a 65.64 ± 1.2 c 7.64 ± 2.4 a

Days L* a* b* Chroma Hue

14 47.08 ± 0.6 a 63.16 ± 0.1 f 11.03 ± 0.7 a 64.12 ± 0.3 d 9.91 ± 0.6 a

Days L* a* b* Chroma Hue

14 47.00 ± 0.9 a 63.34 ± 0.8 e 11.04 ± 1.1 a 64.30 ± 0.9 c 9.88 ± 0.8 a

8.88 ± 1.6 a 5.87 ± 0.1 a 65.00 ± 3.7 c 63.44 ± 0.3 c 7.83 ± 6.2 a 5.30 ± 0.1 a MILD 7ºC 28 42 47.27 ± 0.4 ab 46.61 ± 0.3 a 63.22 ± 0.2 f 62.62 ± 0.2 ef 11.27 ± 0.5 ab 11.96 ± 0.5 b 64.22 ± 0.2 d 63.76 ± 0.1 d 10.11 ± 0.4 a 10.82 ± 0.4 b SEVERE 7ºC 28 42 47.49 ± 1.1 a 47.54 ± 1.2 a 63.33 ± 0.4 e 63.10 ± 0.4 e 10.70 ± 1.2 a 10.84 ± 1.3 a 64.24 ± 0.6 c 64.03 ± 0.6 c 9.58 ± 1.0 a 9.75 ± 1.1 a

8.40 ± 2.8 a 64.34 ± 1.6 c 7.47 ± 2.3 a

16.95 ± 2.5 b 47.37 ± 3.5 b 20.90 ± 1.6 b

56 48.03 ± 0.2 b 62.27 ± 0.2 e 10.67 ± 0.2 a 63.52 ± 0.4 d 9.67 ± 0.1 a

14 56.93 ± 0.7 c 42.28 ± 0.5 d 17.94 ± 0.2 c 45.91 ± 0.5 c 23.00 ± 0.0 c

56 48.10 ± 1.0 a 62.59 ± 0.7 e 10.42 ± 1.1 a 63.46 ± 0.9 c 9.45 ± 0.9 a

14 56.92 ± 1.4 b 42.44 ± 2.1 d 17.45 ± 0.2 b 45.89 ± 2.0 b 22.37 ± 0.7 b

24.68 ± 2.7 c 29.36 ± 1.8 d 36.68 ± 3.4 a 34.99 ± 2.9 a 42.35 ± 0.7 c 56.96 ± 2.8 d MILD 37ºC 28 42 63.95 ± 0.4 d 67.62 ± 0.4 e 27.56 ± 0.5 c 18.87 ± 0.4 b 24.95 ± 0.2 d 30.12 ± 0.5 e 37.18 ± 0.5 b 35.54 ± 0.6 a 42.16 ± 0.3 d 57.93 ± 0.1 e SEVERE 37ºC 28 42 63.59 ± 1.5 c 67.44 ± 1.3 d 27.63 ± 1.6 c 19.46 ± 1.5 b 24.54 ± 0.6 c 29.22 ± 0.5 d 36.96 ± 1.6 a 35.12 ± 1.2 a 41.62 ± 1.0 c 56.37 ± 1.5 d

N=3 ± SD. Different letters means significantly different at P < 0.05 according to Tukey HSD Multiple Range Test.

29

30.84 ± 1.9 d 33.92 ± 2.6 a 65.50 ± 1.8 e 56 69.68 ± 0.4 f 14.18 ± 0.3 a 32.89 ± 0.3 f 35.82 ± 0.4 a 66.68 ± 0.3 f 56 69.48 ± 1.1 d 14.29 ± 1.0 a 32.07 ± 1.0 e 35.11 ± 1.4 a 66.01 ± 0.9 e

1.304 1.401 1.094 LSD P<0.05 0.253 0.187 0.244 0.233 0.196 LSD P<0.05 0.700 0.699 0.551 0.722 0.590

Table 3. Viable plate counts of the microorganisms analyzed (psycrophilic microorganisms and yeast and moulds) of the LM isotonic drink (pH 2.29 ± 0.01) untreated and treated, during the storage time at refrigeration temperature (7ºC).

7°C

THERMAL TREATMENT

Psychrophilic

Mild treatment

Severe treatment

Yeast & moulds

Mild treatment

Severe treatment

SAMPLE

Untreated

0 <10 CFU/mL

14 <10 CFU/mL

Time (Days) 28 <10 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Untreated

<10 CFU/mL

<10 CFU/mL

33 CFU/mL

1 x 106 CFU/mL

> 106 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Untreated

<10 CFU/mL

<10 CFU/mL

2.5 CFU/mL

3.1 x 103 CFU/mL

1.6 x 104 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Untreated

<10 CFU/mL

2.7 x 102 CFU/mL

5.6 x 102 CFU/mL

5.6 x 106 CFU/mL

2.9 x 108 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

30

42 <10 CFU/mL

56 2.6 x 103 CFU/mL

Table 4. Viable plate counts of the microorganisms analyzed (mesophilic microorganisms and yeast and moulds) of the LM isotonic drink (pH 2.29 ± 0.01) untreated and treated, during the storage time at an abusive temperature (37ºC). 37°C

THERMAL TREATMENT

Mesophilic

Mild treatment

Severe treatment

Yeast & moulds

Mild treatment

Severe treatment

SAMPLE

Untreated

0 <10 CFU/mL

14 <10 CFU/mL

Time (Days) 28 <10 CFU/mL

42 <10 CFU/mL

56 <10 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Untreated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

Untreated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

1 x 102 CFU/mL

> 100 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

> 100 CFU/mL

Untreated

<10 CFU/mL

<10 CFU/mL

5 CFU/mL

> 100 CFU/mL

1.7 x 102 CFU/mL

Treated

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

<10 CFU/mL

> 100 CFU/mL

31

Highlights

-

Heat treatments were applied to new isotonic drinks of lemon juice and maqui

-

Colour were affected by heating although drinks retained their red coloration

-

Heating did not affect antioxidant capacity and anthocyanin content and retention

-

Mild treatment with preservation at 7ºC is the ideal to prevent microbial growth

-

Mild heating with shelf life at 7ºC also kept the bioactivity of the isotonic drink

32